Project 2 - Mechanisms of Axon Injury in Ischemic White Matter Damage to cerebral white matter (WM) is an important consequence of stroke, cardiac arrest, and perinatal hypoxia-ischemia. WM injury is also observed in clinical settings associated with disruption of the neurovascular unit and blood brain barrier, such as with chronic hypertension and cerebral amyloid angiopathy. Since central nervous system axons do not regenerate significantly in clinical situations, an important goal for therapy is to interrupt injury pathways before irreversible damage occurs. The initial events in WM hypoxia-ischemia have been well characterized: axon energy deprivation triggers sustained overactivation of voltage-gated sodium channels and subsequent intra-axonal calcium overload. Despite this solid knowledge of early events, there is little understanding of the pathways downstream of calcium accumulation that are required for axon injury. The goal of this project is to discover novel approaches to therapy by identifying mechanisms of irreversible degeneration of myelinated axons in hypoxic-ischemic white matter. We will develop lentivirus vectors, which are injected stereotaxically in adult mouse motor cortex in vivo, to direct selective expression of molecular pathway inhibitors in axons in the corpus callosum. We will first examine effects of transgene expression using a white matter brain slice model which allows functional and structural assessment of YFP-labeled axons during and up to 10 hours after oxygen-glucose deprivation. Aim 1 examines the hypothesis that delayed hypoxic-ischemic axon degeneration requires activation of calpain, caspase, or ubiquitin-proteasome system (UPS) pathways. Aim 2 examines the hypothesis that ischemic axon injury is reduced by prior overexpression of nicotinamide mononucleotide adenylyltransferase (NMNAT1) and other proteins related to the Wallerian degeneration slow mouse mutant. In collaboration with Project 2, Aim 3 examines the hypothesis that ischemic axon injury is reduced by treatments which induce long-term tolerance to gray matter ischemic injury in mice. In Aim 4, approaches found to limit or delay axon degeneration in brain slices will be further tested using murine models of selective WM ischemia (produced by WM injection of the vasoconstrictor, endothelin-1) and combined gray and white matter stroke (produced by middle cerebral artery occlusion). RELEVANCE FOR PUBLIC HEALTH: Approximately 700,000 Americans experience a stroke each year. Almost all of these strokes involve the brain's white matter, which transmits signals between brain and spinal cord areas, but little is known of how the white matter is injured or how to prevent this injury. The goal of this project is to identify injury pathways in axons which suggest approaches for stroke therapy.